1. Field of the Invention
Electrophoresis is a separation method based on different migration rates of dissolved ions in an electric field. When the ions migrate through a gel, the chemical and physical properties of the gel influence migration rates of various ions to varying degrees. Therefore, gel electrophoresis provides a higher resolving power than free solution electrophoresis. A gel also reduces diffusion of separated zones after the electric field has been switched off, facilitating the detection of the zones. Gels of different chemical compositions, physical properties and shapes have been used for electrophoresis, in a variety of different apparatuses, for separating ions as small as nucleotides or as large as chromosomal DNA. In general, there are two types of gel electrophoresis: analytical and preparative. In analytical electrophoresis, the gel is discarded after the detection of separated zones. In preparative gel electrophoresis, after the detection of a target molecule, a gel piece containing the zone of interest (that is the zone containing the target molecules) is further processed to recover the ions from the gel zone. This invention relates to an apparatus and method for preparative electrophoresis, and especially an apparatus for recovering target molecules from a gel that has undergone electrophoresis.
2. Description of Prior Art
Gel electrophoresis is particularly well suited for the separation of biological macromolecules, such as proteins, nucleic acids and charged polysaccharides. Recovery of the separated macromolecules is often needed in many research applications. In industry, gel electrophoresis is routinely used, for example, for the preparation of high purity oligonucleotides (oligos), especially those longer than about 50 base pairs. Currently, no other method can match the degree of purity of product achieved by gel electrophoresis.
Preparative gel electrophoresis can be carried out in a continuous or discontinuous mode. In continuous mode, the separated zones are collected in the order they emerge from the gel. For example, Hjerten et al. (Analytical Biochemistry, 1969, 27:108–129) describe an apparatus in which protein molecules leaving a polyacrylamide gel enter a bed of agarose particles from which they are eluted using a stream of buffer leading to detector and fraction collector. Alternatively, a semipermeable membrane can be placed near the gel end to prevent dilution of the electroeluting molecules in an electrophoresis buffer, as described for example in U.S. Pat. Nos. 5,284,559, 3,888,758 and 3,719,580.
In a discontinuous mode, the separated zones are made visible, or their position can be determined by other means, for example by comparison to markers. Then the gel part of interest is cut out using a suitable means such as a nylon string, needle, scalpel or razor blade. Different methods have been used for recovering the target molecules present inside the cut gel piece, including crushing the gel into small particles followed by incubation to let the desired molecules diffuse out of the gel. Electroelution recovery of target molecules from gel is the preferred method because it is faster and provides higher recovery yields than other methods. The molecules that are electrophoretically driven out of the gel need to be stopped from migrating far into the electrophoresis buffer, as otherwise their concentration would decrease to unacceptably low levels. Many solutions that prevent excessive dilution have been disclosed. For example, U.S. Pat. No. 4,545,888 discloses the use of DEAE paper to adsorb nucleic acids that migrate out of the gel. The use of semipermeable membranes, arranged in different ways, is disclosed for example in U.S. Pat. Nos. 5,527,680, 5,439,573, 5,384,022, 5,340,449, 5,102,518, 4,964,961, 4,877,510 and 4,608,147. The disadvantage of the membranes is that they tend to adsorb electroeluted molecules, reducing the yield. To improve the recovery yield, it is necessary to temporarily change polarity of the electric field in order to release the molecules from the membrane. Another disadvantage is that some molecules denature in contact with a membrane, as pointed out by Hjerten et al. (Analytical Biochemistry, 1969, 27:108–129).
When electroeluting fast migrating molecules from low percentage gels, like DNA from agarose gel, then a brief application of the electric field in a small buffer volume may be adequate (U.S. Pat. No. 4,747,918 to Wassenberg). However, this procedure will not work with the molecules characterized by low migration rates or with high percentage gels. In order to slow down the electroeluted molecules, one can increase the ionic strength of the buffer into which the molecules migrate after leaving the gel. It is well known to those skilled in the art that migration rate of a molecule is reduced when it enters a buffer of high conductivity (concentration). Thus Kragt et al. in U.S. Pat. No. 3,989,612 added sodium chloride to phosphate buffer on one side of their electroelution cassette in which the gel makes a leak proof seal with other parts of the cassette.
In U.S. Pat. Nos. 4,576,702 and 4,576,703 Peck et al. disclose the use of a salt solution pipetted into a reservoir that receives electroeluted molecules. A similar disclosure can be found in U.S. Pat. No. 4,725,348 to Diekmann. In the apparatuses of both Peck et al. and Diekmann, electrode compartments are separated by a block (Diekmann), or a bridge (Peck et al.), that ascends from the bottom of the electrode compartments. The V-shaped reservoir, that contains a salt solution and receives the electroeluted molecules, is constructed in the block (bridge) in such a way that it connects the two electrode compartments. The two electrode compartments communicate also through at least one additional conduit that needs to be closed during electroelution. Prior to removal of the salt solution containing the electroeluted molecules, it is necessary to open other conduits in order to lower the level of electrophoresis buffer below the level of the openings of the V-shaped reservoir. For this purpose, the apparatuses of Peck et al. and Diekmann contain valves that need to be closed and opened in a defined manner, making the operation complicated. In addition, the need for valves inside the apparatus makes the construction of their apparatuses complex and expensive.
Now it has been found that the apparatus of present invention can overcome the above drawbacks of the apparatuses disclosed in prior art because it is simplified and is effective.